DNS
Chain of nucleotides aka polynucleotide
Double helix
5’ to 3’
Gene
Functional unit of genetic information
Located on chromosomes or other genetic elements in cell
Composed of DNS
DNa bases (pyrimidine)
Cytosine: DNa and rna
Thymine: Dna only
Purine bases(dna)
Adenine and guanine: both dna and rna
Watson and crick
Proved DNa had double helix structure with sugar and I hospital groups with nitrogen bases.
Wilkins and Rosalind Franklin
Were a huge part of Dna helix discoveration, Franklin used x rays diffraction images
the double helix
All cells Bacteria:millions of nucleotides Two strands are anti palRallel A-t , g - c High heat breaks hydrogen bonds, gc rich melts at higher temp them at
Size=length
Measured in base pairs 1000 base pairs = 1kbp 1000000 base pair is mega base pair E. coli has 4.64 Mbps .34nm along helix, ecoli is 1 mm
Genetic information flow
Central dogma of molecular bio: dna>rna>protein
Replication
Dna is duplicated
Transcription
Information from dna is transferred to mRNA…NOTE bottom strand
Translation
Information in rna builds proteins
Supercooled dna
Further twisted to save space
Negative :unwound
Positive:overwound
Relaxed dna..number of turns predicted by number of base pairs
Dna gyrate
One part of circle is laid over other
Helix makes contact in two places
Unbroken helix is passed through the break
Following dna gyrate activity two super coils result
Genome
Entire complement of genes in cell or virus
Chromosome
Main genetic element in prokaryotes
encoding Core functions
Horizontal gene transfer equation
N=No X 2^n
Horizontal transfer
Transfer of genetic material from one bacterial cell to another , related or not related
Antibiotic resistance :1950
Today:who,cdc: a health crisis…post antibiotic era
Three mechanisms
Transformation
Conjugation
Transduction
Conjugation
Transfer of dna from a donor to recipient after cell contact via plasmid
Transduction
Transfer of dna between bacteria via bacteriophages
Transformation
Uptake of naked dna
Plasmids
Replicons,circular or linear, replicate separately from chromosomes
Mobile genetic mosaics that spread multiple beneficial traits,drug resistance,virulence…important in bacterial evolution and adaptation
Most bacteria have a lot of them
Hi vs low copy number , self transferable, difference in host range
Reason for Increase in resistance
In gram neg bacteria is due to mobile genes in plasmid
Virulence
Encoded in plasmids Enables pathogen to colonize Enables pathogen to damage host Hemolysis Enterotoxin
Transposable elements
Segment of dna that can move from one site to another site on same or different dna molecule
Types:insertion sequences,transposing, special viruses
Viruses
Contain either dna or rna genomes
Linear or circular
Single or double stranded
E. coli chromosome
Lab strain: 4.64 Mbps
0157: h7 5.5 mbp
40 percent of predicted proteins are unknown function
Average protein: 300 amino acids=900 bp=1kbp
Semi conservative (dna replication)
Each of two double helices have one parental and one new strand
Replication always goes from 5 triphosphate to 3
DNA polymerase
Catalyze the additions of dntp
5 different polymerases in ecoli primary is polymeraeIII
DNA POLYMERASES require primer from rna
Replication fork
DNA synthesis begins at origin of replication in prokaryotes:oriC
Fork is zone of unwound DNA where replication occurs
DNA helices unwinds the DNA
Connecting DNA fragments in lagging strand
- syhnthesis on lagging strand continues until it reaches previous synthesized DNA
- DNA polymerase 1 removes rna primer and replaces with DNA
- dna ligase seals nick in DNA
Bidirectional DNA synthesis in prokaryotes
Two replication forks moving in opposite directions
Replisome
Complex of multiple proteins involved in replication
DNA is pulled through replisome
Accuracy of DNA replication
Extremely accurate due to proofreading
Errors are extremely rare
Polymerase detect mismatch through incorrect hydrogen bonding
Polymerase chain reaction (pcr)
1, add DNA template primers, DNA polymerase
Heat and cool
Heat and cool
Applications of pcr
Surveying different groups of environmental organisms,s
Cloning DNA into plasmids
Identifying specific bacteria
Looking for a specific gene
Transcription process
Carried out by rna polymerase
Uses DNA as template
Rna precursors are atp,gtp,ctp,utp,
Chain growth is 5 tom3 same as replication
Only one of two strands is transcribed by rna
Promoters
Site of initiation of transcription
Transition terminators
Site where transcription stops
Operon
A group of related genes cotta scribed on a polycistronic(encoding group) mrna
Exons and introns
Exons are the Coding sequence
Introns are the intervening sequences
Introns rare in archaea
Eukaryotic rna processing
Many rna molecules are altered before they carry out their role in the cell
Rna splicing
Takes place in nucleus
Removes introns from rna transcripts
Performed by spliceosome
Rna capping
Addition of methylated guanine to 5 end mRNA
Poly a tail
Addition of 100-200 adenlylane residues
stabilizes mRNA and is required for translation
Proteins
Play major role in cell functions
Catalytic proteins(enzymes)
Structural proteins
Proteins: polymers of amino acids
Polypeptide
Amino acids linked together y peptide bonds
Primary structure
Linear array of amino acids in a polypeptide
22 amino acids
Genetic code
Triplet of nuclei acid bases (codon) encodes a single amino acid
Specific codons for stopping and starting translation
Stop codons
Uaa uag uga
Start codon
Aug
Open reading frame (orf)
Aug followed by codons and a stop codon in same frame
Anticodon
Three bases on tRNA that recognize codon on mRNA
Wobble irregular base pairing allowed at third position of tRNA
Transfer tRNA
Atleast one tRNA per amino acid
Bacterial cells have 60 different tRNA
Mammalian cells have 100-110
Specific for both codon and its cognate amino acid
Arp is required to attach amino acid to tRNA
Ribosomes
Site of protein synthesis
Thousands per cell
Combination of rRNA and protein
E. coli has 52 distinct proteins
Translation steps
initiatione:two ribosomal subunits assemble with mRNA
Elongation:amino acids are brought to the ribosome and are added to the growing polypeptide , occurs in a and p sites(translocation)
3.termination: occurs when ribosome reaches stop codon,release factors.
Polyamory
A complex formed by ribosomes simultaneously translating mRNA
Antibiotics
Inhibit translation by interacting with ribosomes
Desaturation
When proteins are exposed to extreme heat,ph,or certain Chemicals
Results in loss of biological properties(microwaves)
Signal sequences
Found on proteins requiring transport from cell
Prevent protein from folding
Found at beginning of protein molecule
Genome
Complement of genetic information
Includes genes, regulatory sequences , and non coding DNA
Includes chromosome and plasmids
Genomics
Discipline of mapping, sequencing, and analyzing , and comparing genomes
Sequencing
Determining precise orders of nucleotides I’m a DNA or rna molecule
Illumina sequencing
Lucifer are consumes arp and emits light
Light flash detected by sensor
Pacific biosciences smrt
Single molecule real time sequencing
Reactions carried out in nano containerssingke stranded DNA fragments attached
Complementary strand synthesized our fluorescent tags attached
Shot gun sequencing
Much of sequencing is redundant
Computer algorithms are used
Closed vs draft genome
Closed genome relies on manpower
More expensive
More information
Bioinformatics
Science that applies powerful computational tools to DNA and protein sequences
Annotation
Converting raw sequence data into a list of genes present in the genome
Majority of genes encode proteins
Genomes of organelles
Mito and chloroplasts contain a small genome
Also contain the necessary machinery for protein synthesis
Chromosomal islands
Region of bacterial chromosome of foreign origin that contains clustered genes for some extra property such as:
Virulence
Symbiosis
Metagenome
The total gene content of the organisms present in environment
Major modes of regulation
- Controls the activity of preexisting enzymes
Post-translational regulation, very rapid process
2.other controls amount of enzyme
Regulates transcription,translations…slower process
DNA binding proteins
mRNA transcripts have short half life
Regulation requires protein binding to DNA
Small molecules influence binding of regulatory proteins to DNA
Outcomes for DNA binding
- Catalyze a specific reaction. On DNA molecule
- The binding event can block transcription
- The binding event can activate transcription
Control of transcription/translation
Resulted by Interactions between small molecules and DNA binding proteins
Negative control
A regulatory mechanism that stops transcription
Repression
Induction
Repression
Preventing the synthesis of an enzyme in response to a signal
Affects anabolic enzyme
No wasted energy
Induction
Production of an enzyme in response to a signal
Affects catabolic enzymes
No wasted energy
Inducer
Substance that induces enzyme synthesis
Corepresser
Substance that represses enzyme synthesis
Effectors
Collective term for inducers and repressors
Affect transcription indirectly by binding to specific DNA binding proteins
Operon
Cluster of genes arranged in A linear fashion whose expression is under control of single operators
Downstream of promoter
Transcription is physically blocked when repressors bind to operator
Iptg
Induces beta galactosidase but not hydrolyzed by enzyme
Positive control
Regular protein activates the binding rna of polymerases to DNA
Maltose catabolism in E. coli
DNA sequence: activator-binding site
Activator protein
Helps rna polymerase recognize promoter
Catabolism repression
Example of global control
Diauxic growth
Two exponential growth phases
Catabolite repression
Transcription is controlled by an activator protein
Activator protein
Cyclic amp
Key molecule In many metabolic control systems
Quorum sensing:
Mechanism by which bacteria assess their population density
Discovered as mechanism regulating light production
Regulatory rna molecules
Exert their affects by base pairing with mRNA
Capsid
Protein she’ll that surrounds the genome of a virus particle
Capsomere
Subunit of the capsid
Nucleocapsids
Constructed in highly symmetric ways
Helical symmetry…rod shaped viruses
Lysozyme
Makes hole in cell wall
Lyses bacterial cell
Neuraminidases
Degrades silicon acid, helps virus assembly
Temperate viruses
Can undergo a stable relationship within the host but can also kill cells through lyric cycle
Lysogeny
State where most virus genes are not expressed and virus genome is replicated in synchrony with host chromosome